Uniform TitleStructural studies of HIV-1 reverse transcriptase: resistance to AZT via ATP-mediated excision
NameTu, Xiongying (author), Arnold, Eddy (chair), Sarafianos, Stefan (internal member), Olson, Wilma (internal member), Jones, Roger (internal member), Hughes, Stephen (outside member), Rutgers University, Graduate School - New Brunswick,
SubjectChemistry and Chemical Biology,
DescriptionAZT, 3'-azido-2',3'-deoxythymidine, is a widely used anti-AIDS drug. Incorporation of AZTTP, the triphosphate form of AZT, into a growing DNA chain by the human immunodeficiency virus (HIV) reverse transcriptase (RT) blocks chain elongation. HIV develops resistance to AZT by acquiring mutations in reverse transcriptase (RT) that enhance ATP-mediated excision of AZTMP. This excision frees the end of the primer strand, making it possible for RT to continue viral DNA synthesis. The product of the excision reaction is AZT adenosine dinucleoside tetraphosphate (AZTppppA). In order to understand the structural bases of this resistance mechanism, five different HIV-1 RT complex structures involved in this ATP-mediated excision were determined using a cross-linking strategy and X-ray crystallography.
The structures of wild-type and AZT-resistant HIV-1 RT with a dsDNA and an AZTppppA (excision product complex) were determined to 3.15 and 3.2 Å resolution, respectively. Superposition of these two structures reveals that the ATP binds differently to wild-type RT and AZT-resistant RT. Primary resistance mutations do not improve a pre-existing site but create a new, higher-affinity binding site for ATP. The primary AZT-resistance mutations K70R and T215Y make significant contributions to ATP-binding, whereas, secondary resistance mutations assist the binding of ATP to RT. These structures also provide insights into antagonism between AZT-resistance mutations and other nucleoside-analog resistance mutations, including K65R and K70E.
The structure of AZT-resistant HIV-1 RT with a pre-translocation AZTMP-terminated DNA was determined to 3.7 Å. This structure has the fingers subdomain in a closed configuration instead of an open configuration found in the reported structure of the wild-type HIV-1 pre-translocation complex. The pre-translocation complex with the fingers in a closed configuration may be the preferred acceptor for ATP. This structure also implies polymerization/pyrophosphorolysis may occur without major opening of the fingers.
The structures of the AZT-resistant HIV-1 RT with a post-translocation AZTMP-terminated DNA and the unliganded AZT-resistant HIV-1 RT were determined to 2.9 and 2.65 Å resolution, respectively. Structural comparisons of these two complexes and excision product complexes suggest that the side chains of the two primary resistance mutations may undergo conformational changes that should be induced by the ATP.
NoteIncludes bibliographical references (p. 236-245).
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.